O-acetyl thymotic acid anhydride and preparation thereof



3,l6fi,58b Patented Jan. 19, 1965 O-ACETYL THYMOTIC ACID ANHYDRIDE AND PREPARATION THEREOF Piero Maria Carrara, 36 Col-so Monfo'rte, Milan, Italy N Drawing. Filed July 24, 1964, Ser. No. 385,064

3 Claims. (Cl. 260-479) Thymotic acid, or 2-hydroxy-3-isopropyl-6-methylbenzoic acid has been long known. It can be prepared according to the indications in literature, by a modification of Kolbes synthesis; compare e.g. Gazz. Chim. Ital. 39, 325 (1911) or US. Patent No. 2,824,892 dated February 25, 1958.

Various authors attribute to thymotic acid properties generally similar to those of salicylic acid; more particularly it is disclosed as having an antipyretic and antiseptic activity. Recently its analgesic activity was emphasized. However, it was likewise ascertained that thymotic acid is of a considerably higher toxicity than salicylic acid and its derivatives.

It has now been found in the course of investigations which lead to this invention that thymotic acid also possesses anti-edema and anti-granulous activities decidedly higher than those of preparations known heretofore, employed in the therapy of rheumatic diseases, such as salicylic and butazolidine preparations. It is true that, in respect to antigranulous activity, salicylic and butazolidine preparations excel, the weight required being the same as with cortisone preparations, but it was also found that the latter are practically analgesically ineiiicient and are moreover objectionable in various respects in that they detrimentally affect the hydrosalinebalance of the human individual.

More particularly, this invention provides the O-acetyl thymotic acid anhydride (AAT) of the structural formula:

administration, was found to exceed 1500 mg./kg., an.

extraordinarily high value as compared with 500 mg/kg. of aspirin or 160 nag/kg. of sodium thymotate or even of the 175 rug/kg. of choline thymotate.

The following table summarizes in succinct and conventional manner the activities of the anhydride 0 O-acetyl thymotic acid:

Antirheumatic activity Analgesic activity Anti-edema Anti-granuloma AAT Reliable Reliable Reliable. Acetylsalicyclic acid. Significant, apparently Significant, apparently Significant, apparently not not reliable. not reliable. reliable. Diphcnylbutazone Significant. apparently Quite reliable Significant, apparently not not reliable. Y eliable. Prerlnisnlnne Nil Significant, apparently Reliable.

not reliable.

" motic acid anhydrides of the general formula:

I O O CH3 CH3 wherein R is a linear or branched chain aliphatic radical having four carbon atoms at the utmost, or a substituted or non-substituted aralkyl or phenyl, and wherein R is a linear or branched chain aliphatic radical having four carbon atoms at the utmost, or a substituted or nonsubstituted phenyl.

The preparation of anhydrides according to this invention necessitates a special operational cycle which is not disclosed or suggested by the literature. More particularly, the reaction conditions of the various reactants should be predetermined and adjusted to obtain first the anhydride of thyrnotic acid which is successively acylated to yield the O-acyl-thyrnotic acid anhydrides.

The reverse operation, consisting in acylating thymotic acid to O-acyl thymotic acid and converting the latter to its anhydride in accordance with methods known in the art, does not lead to any formation of the abovementioned O-acyl thymotic acid anhydride.

So, e.g., in order to obtain AAT, thymotic acid is convented to its corresponding anhydride by reaction with acetic anhydride. If desired, the two reactants can be admixed with an anhydrous solvent, such as benzene. However, in order to secure a satisfactory output it is important to provide excess acetic anhydride, i.e., a proportion of at least 5 parts by weight to 1 part thymotic acid. The optimum range is 5-10 parts by weight.

The mixture of the two reactants is refiux-reacted at a temperature ranging between and C. during a period ranging between 1 and 4 hours which can in any case be experimentally determined to establish its correct value. The resulting product contains the anhydride of thymotic acid which according to this invention shall now be acylated. For this purpose unreacted acetic anhydride is first evaporated by operating at atmospheric pressure or subatmospheric pressure. The residue is reacted with a large excess of glacial acetic acid, by a proportion of at least 3 parts by weight to 1 part of the residue at a temperature ranging between 70 and 80 C., whereupon the acetic solution is cooled to room temperature. AAT is separated by dilution of the acetic solution with water. This dilution should be carried out with special care. First of all, the volume of the dilution water should not exceed the volume of the acetic solution. Secondly, dilution should be carried out gradually. while constantly stirring. Under these conditions, the precipitation of AAT starts and is substantially complete after about 24 hours standing.

It will be obvious that the invention covers acylating with other acylating agents, such as with the chloride of the acid to be introduced into the molecule of the thymotic acid, such as benzoyl chloride, cinnamoyl chloride and the like. The respective processes will be obvious from the folowing specific examples.

EXAMPLE 1 100 g. thymotic acid are suspended in 600 g. acetic anhydride diluted with 1 liter anhydrous benzene. The resulting mixture is heated and gently reflux-boiled, the temperature being gradually raised to 130150 C.

After about 3 hours the unreacted acetic anhydride is distilled in vacuum, the residue being dissolved at 7080 C. in 400 ml. glacial acetic acid. The acetic solution is cooled to room temperature and slowly admixed with 100 ml. water while vigorously stirring. This starts the separation of a white precipitate. 300 ml. water are then added at a quicker rate still vigorously stirring, whereupon the solution is allowed to stand during 24 hours.

The resulting precipitate is collected, washed with 50% acetic acid and recrystallized from ethyl alcohol at 95 C. The result is about 40 g. white crystalline product having a melting point of 103-104 C.

Analysis.--Calculated for C H O C, 68.72%; H, 6.61%; O, 24.67%. Found: C, 68.54%; H, 6.44%; O, 24.81%.

The product is saponified with N/ 1 caustic potash and is found to be of 99-100% purity, when employing an eqivalent equal to 113.5 (molecular weight 454:4). Titration as anhydride according to D. M. Smith and W. M. D. Bryant (JACS 58, 2452 (1936)) confirms the above-mentioned equivalent corresponding to the molecular weight of AAT.

EXAMPLE 2 100 g. thymotic acid are suspended in 900 g. acetic anhydride. The reaction mixture is maintained during 2 hours at 1l0-120 C. by reflux. The acetic anhydride is then distilled in vacuum, operation being further carried out as in Example 1.

45 g. AAT are obtained.

EXAMPLE 3 100 g. thymotic acid are reflux-reacted with 300 ml. thionyl chloride. On completion of reaction the unreacted thionyl chloride is eliminated by distillation, the residue being taken with 200 g. acetic anhydride. The solution is then maintained during 2 hours at llO-120 C., whereupon the acetic anhydride is distilled in vacuum.

Further operation is carried out as in Example 1. 60 g. AAT are obtained.

EXAMPLE 4 100 g. thymotic acid are reflux-reacted with 200 g. thionyl chloride. After distillation of the unreacted chloride, the residue is suspended in water and treated by small batches with benxoyl chloride and sodium bicarbonate, in order to maintain the solution near neutrality.

A substance is obtained which, upon recrystallization from ethyl alcohol, is found to be the anhydride of O-benzoyl-thymotic acid.

EXAMPLE 5 g. thymotic acid are converted into the bisodium salt in an anhydrous state. This salt is suspended in 500 ml. benzene and admixed with 200 g. thionyl chloride, whereupon the mixture is reflux-reacted during 2 hours.

The cooled mixture is subsequently filtered from the sodium chloride separated during the reaction, the solvent (benzene) being distilled and the residue being treated similarly to that in Example 4, but with cinnamoyl chloride.

A substance is obtained which, upon recrystallization from ethyl alcohol, is found to be the anhydride of O- cinnamoyl thymotic acid.

The utility of O-acetyl-thymotic acid anhydride (AAT) in the treatment of rheumatoid arthritis (osteoarthritis, acute rheumatism, bursitis and lumbago was investigated by clinical studies upon 28 patients. 15 of these patients suffered from rheumatoid arthritis, 5 from osteoarthritis, 3 had an acute rheumatism, 1 a bursitis, and 4 a lumbago. Every patient was subjected to a general examination and a history record on admittance. Liver function tests, urinalysis, hematocrit and erythrocite sedimentation rate determinations were effected in every case prior to the start of the therapy. A general examination was repeted in every case at least once a week after the starting of the therapy. Urinalysis, hematocrit and erythrocite sedimentation rate were determined at two weeks interval and liver function test at least at the end of the treatment trial.

Every patient received 1 tablet orally 3-4 times daily containing 250 mg. AAT each, except for 7 patients selected randomly in the rheumatoid arthritis group, which received the same amount of identical tablets but containing placebo.

Disease activity In the rheumatoid arthritis group this was measured before and after the course of treatment according to the following criteria:

(1) Pain on digital pressure over the affected joint, measured in four grades:

0--No pain 1Slight pain 2Severe pain (with wincing) 3Pain (with wincing and withdrawal) (2) Functional capacity as adopted by Duthie and Coll.,

Ann. Rheum. Dis. 14, 133 (1954):

1-Normal activity 2-Moderate restriction 3--Marked restriction 4-Confined to chair or bed (3) Erythrocite Sedimentation Rate (mm./hr., Westergren). (4) Swelling of the affected joint, measured in three grades:

1--No swelling 2-Slight swelling 3-Marked swelling (5) Patients subjective opinion on his conditions, graded as follows:

0-Unchanged lWorse -2Much worse +1Somewhat improved +2-Greatly improved (6) Overall results were expressed as follows: tration for periods over to 14 weeks with daily dosages Very good of 750-1000 mg. orally. Good (6) In no case were changes detected at urinalysis; Moderate hematocrit and liver function tests, nor evidence of Poor gastrointestinal bleeding was reached.

TABLE I Grouping 0f the rheumatoid arthritis patients Duration 7 Treatment V Case Sex Age of disease Previous treatment Overall Side No. (years) results efieets Drug Duration M 46 4 None AAT 13 weeks"... Good M 47 6 Aspirin cortisone chloroqulne Itching. F 44 3 Cortisone chloroquine M 52 7 Oortisone Good F 56 8 Cortisone chloroquine Moderate.-- Do. F 51 4 Aspirin- 11 weeks Good F 43 2 Norm weeks Very good M 57 5 Chloroquine AAT 14 weeks-.- Good M 52 5 Cortisone aspirin F 41 3 None F 47 6 Cortisone chloroquine... F 44 4 Cortisone F 59 9 Cortisone chl M a 60 8 Cortisone Do M 43 2 dn Similar criteria have been followed'for the evaluation TABLE II of the eifect of AAT therapy in the miscellaneous group. Results are summarized in the following tables:

Table IGrouping of the rheumatoid arthritis patients.

Clinical particulars of the rheumatoid arthritis group at the start and at the end of course Table IIClinical particulars of the rheumatoid arthritis i g Erythmcite group t th t t d t th d f course Case No. Pain capacity Swelling Sedinfiergtation Subjective Table III-Comparison of AAT and placebo efiects in a e the rheumatoid arthritis group.

2-0 2-1 1 2 2 Table IV-Groupmg and results in the miscellaneous H group 7 I '7 2 0 3 1 1 0 25 18 2 Table VOverall results of AAT therapy related to the 4 2-2 H) 24-22 11 2-1 -0 31-26 2 different diseases. To uty g 1 1:8 33 xi 3-3 1-1 1-0 27-25 0 The administratlon of AAT was well tolerated by alg-g a 0 3 +l most'all of the 21 patients who received the drug. 9:? Side eifects appeared very seldom and in no case dis- 3:? 1 52-35 continuation of the course was necessary. H H 3:

In no case were changes detected at the urinalysis,

hematocrit and liver function tests determination. In most cases stool examination was eifected during and at the end of the course for the detection of gastrointestinal bleeding but the benzidine reaction was always negative. The few cases of side eifects are recorded in Tables I and 1V.

From these tests it is concluded that:

(1) The oral administration of 750-1000 mg. daily of AAT for periods ranging from 6 to 14 weeks had an TABLE III appreciably favorable effect on the disease activity of patients suffering from rheumatoid arthritis, osteoarthritis, acute rheumatism, lumbago.

(2) The administration of placebo to a control group Compaison of AAT and placebo effects in the rheumatoid arthritis group of rheumatoid arthritis patients induced favorable ef- Number 08505 fects of a consistently lower degree and in a lower number of cases. This favorable effect of placebo Impmved Unchanged worsened administration has already been evidenced by many clinicians. mam g "5 (3) The effect of AAT administration seems to be more Function- 5 3 evident in subjects which received no previous therapy Swelling g and when the duration of the disease was shorter at 3 4 the time of the course starting. ESR AAT 2 (4) Among the symptoms considered, pain and subjecu i t v tive state seem to be the more favorably affected. OveralL 7 1 (5) The toxicity of the drug appeared to be very low 2 4 and no major side etfects were induced by its adminis- TABLE IV Grouping and results in the miscellaneous group Therapy Case N 0. Age Sex Diagnosis Result Side efiects Drug Duration 16 M Acute rheumatism- AAT 6 weeks Good 49 M Osteoarthritis AAT Weeks Moderate- 47 F o 8 weeks 42 M Lumbago 6 weeks 27 F Acute rheumat 58 F Lumbago 62 F Osteoarthritis. 12 weeks. Dizziness. 37 M Bursitis 6 weeks- 57 M Osteoarthritis 8 weeks.-- 31 M Acute rheumatism. 6 weeks 55 M Ostcoarthrisis 10 weeks 48 M b 6 weoks Moderate... Itching. 57 F 10 wceks Good TABLE V 2. The method of preparing the anhydride of O-acetyl Overall results of AAT therapy related to the dz'fierent diseases Number of Cases Improved Unchanged Rheumatoid arthritis 6 2 Osteoarthritis 4 1 Acute rheumatism. 2 1 Lumbago 4 Bursitis 1 This is a continuation-in-part of application Serial No. 142,009, filed October 2, 1961, now abandoned.

What is claimed is:

1. The anhydride of O-acetyl thymotic acid as represented by the following structural formula:

thymotic acid, comprising the steps of reflux reacting thymotic acid with an excess of acetic anhydride at a temperature of between 100 and 150 C. for a period of time between 1 and 4 hours, subsequently evaporating from the reaction product the unreacted acetic anhydride, reacting the residue by dissolving said residue in at least three parts of glacial acetic acid per part of said residue at a temperature between about and C., and precipitating the O-acctyl thymotic acid anhydride from the glacial acetic acid solution by dilution of said glacial acetic acid solution with water in a volume not exceeding that, of said glacial acetic acid solution, said water being added gradually with constant stirring.

3. The method of claim 2 wherein said thymotic acid is refluxed reacted with at least about five parts by weight of said acetic anhydride per part of thymotic acid.

References Cited by the Examiner UNITED STATES PATENTS 2,236,125 3/41 Wiezevich 260-480 2,731,492 1/ 5 6 Kamlet 260-480 FOREIGN PATENTS 9,898 1897 Great Britain. 478,004 10/51 Canada.

LORRAINE A. WEINBERGER, Primary Examiner. 

1. THE ANHYDRIDE OF 3-ACETYL THYMOTIC ACID AS REPRESENTED BY THE FOLLOWING STRUCTURAL FORMULA:
 2. THE METHOD OF PREPARING THE ANHYDRIDE OF O-ACETYL THYMOTIC ACID, COMPRISING THE STEPS OF REFLUX REACTING THYMOTIC ACID WITH AN EXCESS OF ACETIC ANHYDRIDE AT A TEMPERATURE OF BETWEEN 100* AND 150*C. FOR A PERIOD OF TIME BETWEEN 1 AND 4 HOURS, SUBSEQUENTLY EVAPORATING FROM THE REACTION PRODUCT THE UNREACTED ACETIC ANHYDRIDE, REACTING THE RESIDUE BY DISSOLVING SAID RESIDUE IN AT LEAST THREE PARTS OF GLACIAL ACETIC PER PART OF SAID RESIDUE AT A TEMPERATURE BETWEEN ABOUT 70* AND 80*C., AND PRECIPITATING THE O-ACETYL THYMOTIC ACID ANHYDRIDE FROM THE GLACIAL ACETIC ACID SOLUTION BY DILUTION OF SAID GLACIAL ACETIC ACID SOLUTION WITH WATER IN A VOLUME NOT EXCEEDING THAT OF SAID GLACIAL ACETIC ACID SOLUTION, SAID WATER BEING ADDED GRADUALLY WITH CONSTANT STIRRING. 